SCOTTSDALE POLICE DEPARTMENT FORENSIC SERVICES …

SCOTTSDALE POLICE DEPARTMENT FORENSIC SERVICES DIVISION-

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Blood Alcohol Analysis Procedures Manual

Original Adoption Date: 2-22-10 Version: PM-TOX-001.1 Version Effective Date: 7-11-12 Issuing Authority: Kris Whitman, QAM

Toxicology Discipline Procedure Manuals Blood Alcohol

1. Introduction ....................................................................................................................................... 2

2. Personnel ........................................................................................................................................... 2

3. Evidence Control.............................................................................................................................. 2

4. Validation ........................................................................................................................................... 3

5. Analytical Procedures..................................................................................................................... 5

6. Equipment Calibration and Maintenance................................................................................... 9

7. Reports ............................................................................................................................................. 11

8. Safety................................................................................................................................................. 13

9. Proficiency/Competency Testing............................................................................................... 13

10. Outsourcing..................................................................................................................................... 13

11. Glossary ........................................................................................................................................... 14

12. References.....................................................................................................13 13. Appendices

13.1 Standard solution preparation...................................................................14 13.2 Quality review and criteria for analytical data................................................14 14. Abbreviations................................................................................................ 17 15. Revision History ........................................................................................................................... 18

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SCOTTSDALE POLICE DEPARTMENT FORENSIC SERVICES DIVISION-

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Blood Alcohol Analysis Procedures Manual

Original Adoption Date: 2-22-10 Version: PM-TOX-001.1 Version Effective Date: 7-11-12 Issuing Authority: Kris Whitman, QAM

1. Introduction

1.1. Goals/objective/scope

This is Scottsdale Police Department Crime Laboratory's procedure for the analysis of ethanol in biological fluids or other liquid matrices using headspace gas chromatography with dual capillary columns and flame ionization detectors. One column is used for quantification, while the other is used as the confirming column. 2. Personnel

2.1. Refer to the QSM for personnel specifications.

3. Evidence Control

3.1. This section will be used to address:

3.1.1. Sample identification (unique number) /labeling All blood vials or other items of evidence for headspace gas

chromatographic analysis submitted by the Scottsdale Police Department will have a unique item number assigned by the ILEADS system before reaching the laboratory. This number will be used to refer to specific test items and their results.

Blood vials or other items of evidence for headspace gas chromatographic analysis submitted by other agencies may not have a preassigned unique number for each item. If this is the case, the analyst will sub itemize the received item and give a unique number to the item(s) to be tested. This number will be used to refer to that specific item and its result(s). 3.1.2. Chain of custody See QSM 5.8.1.1 for general chain of custody procedures. This applies to submissions from all agencies. 3.1.3. Sample handling 3.1.3.1. General. See QSM 5.8.4 for general sample handling procedures. In

addition, all samples for analysis in the Toxicology section will be maintained in a refrigerated condition at any time that access is not required for testing. 3.1.3.2. Biological Sample Safety. Disposable plastic apron and/or other barrier cover(s), single or double disposable gloves, face shield or disposable mask, along with eye protection will be worn when working with blood or other biological samples. All transferring of any potentially hazardous raw biological samples from one vial to another will be performed under a safety hood. All disposable protective clothing and used headspace vials containing blood samples will be disposed of by placing them in the biological waste container which in turn will be removed from the lab on a regular basis for proper disposal. 3.1.4. Sample storage (long term and short term) Short term storage of test items in the Toxicology section may be in the refrigerator inside the evidence intake area or in the refrigerator inside the laboratory.

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SCOTTSDALE POLICE DEPARTMENT FORENSIC SERVICES DIVISION-

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Blood Alcohol Analysis Procedures Manual

Original Adoption Date: 2-22-10 Version: PM-TOX-001.1 Version Effective Date: 7-11-12 Issuing Authority: Kris Whitman, QAM

For long term storage, items will be returned to Property and Evidence or the submitting agency. 3.1.5. Sample security. Samples will be handled consistent with good forensic practice. Samples may only be left unattended in a secure area, i.e. the toxicology laboratory or evidence vault. If a visitor or service technician is present, samples must be attended or locked in a secure area. Laboratory security is as outlined in QSM 5.3. 4. Validation 4.1. This section will be used to address: 4.1.1. Method validation Only the established, validated method will be used for analysis for ethanol by headspace gas chromatography. Other volatiles may be added to the current method or the parameters may be changed to accommodate other volatiles. Refer to QSM 5.4 4.1.2. How method/procedure modifications or variations during testing will be handled. 4.1.2.1. Nonconforming work. There are times when the exact analytical protocol cannot be followed. In these cases the resultant casework is considered `nonconforming' testing. Nonconforming testing is not inherently incorrect; it merely falls outside the bounds of the standard protocol. In the event that nonconforming testing is to be undertaken, it requires preapproval from the Toxicology Technical Leader or, in the absence of the Toxicology Technical Leader, the Quality Manager. A written memo explaining the nonconforming test proposal will be submitted and approved prior to the release of results for nonconforming work. Work itself may proceed on verbal approval from the Technical Leader or, in the absence of the Toxicology Technical Leader, the Quality Manager or their designee. An example of nonconforming work is when, in the opinion of the analyst, an insufficient sample volume is received for duplicate testing according to the regular sample preparation instructions. This can be addressed by performing a single test, manually pipetting the blood sample for replicates, or diluting the blood sample and analyzing replicates of the diluted sample. A copy of the approval memo will go in the case file and also be filed with the Quality Manager. 4.1.2.2. Minor method modifications. Minor method modifications may be required on an infrequent basis due to changes in instrument performance as equipment ages. These modifications may be made by the Technical Leader as necessary and the instrument will be tested with at least one set of standards prior to and after the modification to determine efficacy. If appropriate, the changes will be made to the SOP and a memo will be

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SCOTTSDALE POLICE DEPARTMENT FORENSIC SERVICES DIVISION-

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Blood Alcohol Analysis Procedures Manual

Original Adoption Date: 2-22-10 Version: PM-TOX-001.1 Version Effective Date: 7-11-12 Issuing Authority: Kris Whitman, QAM

generated and signed by members of the section to indicate that they are

aware of the modification(s) and will follow the amended protocol.

4.1.3. Validation of new lots of testing kits

4.1.3.1. Internally prepared standards

4.1.3.1.1 Calibrators. Newly prepared ethanol calibration solutions will be verified by establishing that an acceptable calibration curve can be achieved using the new standard. The calibration must have an R2 0.995. Calibrators will additionally be verified by analyzing a series of known, externally prepared, NIST traceable standards covering the span of the calibration curve against the newly established calibration curve. All known standard calculated values must be within ? 3% of their true values or 0.003 g/dL, whichever is greater, for the calibrators to be acceptable for use. Calibrators may also be sent out to another crime laboratory for

verification of accurate preparation.

Validation information for calibrators will be kept in the "Calibrator

Verification" book.

4.1.3.1.2. Internal standard. Newly prepared internal standard (ISTD) solutions will be evaluated by preparing and analyzing a blank sample using 2500?l of the ISTD and demonstrating absence of any interfering compounds and an area count within ? 20% of the current ISTD lot. The individual preparing the ISTD is responsible for testing that lot of ISTD in duplicate and ensuring that it meets the criteria. The chromatograms and examiners initials and date prepared will be maintained in the Blood Alcohol "ISTD Verification" binder. If the internal standard does not meet this requirement, it will be discarded and reprepared. If there has been a significant change to the method or instrument such that this criterion cannot be met, it will be explained in the maintenance log and on the ISTD log.

4.1.3.1.3. Mixed standards. The mixed standard is to establish qualitatively the ability of the

method to separate a variety of volatile components which may be reasonably expected to appear occasionally in a blood sample from a living human. Quantitative accuracy of the preparation is not required; however the prepared mix may be verified for retention time against an externally prepared mixed standard to ensure presence of all components. 4.1.3.2. Externally acquired standards

All externally acquired control standards in water or whole blood

matrix will be validated before use by analyzing in duplicate against the

established calibration curve.

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SCOTTSDALE POLICE DEPARTMENT FORENSIC SERVICES DIVISION-

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Blood Alcohol Analysis Procedures Manual

Original Adoption Date: 2-22-10 Version: PM-TOX-001.1 Version Effective Date: 7-11-12 Issuing Authority: Kris Whitman, QAM

Analyzed concentrations must be within ? 3% or 0.003 g/dL, whichever is greater, of the target value (supplied by the manufacturer) for the standards to be put in to use. Verifications of this validation will be maintained in the "QC Verification" book.

Externally acquired standards are supplied with an expiration date by the manufacturer. Exceeding this date does not necessarily render the standard unusable or invalid. If a standard is used past the expiration date it must be analyzed in the same manner it was initially validated (in duplicate against a valid calibration curve) as a closing verification when it is removed from use. This will be maintained in the "QC Verification" book along with the initial information. Standards which are removed from use prior to the manufacturer supplied expiration date are presumed to remain valid. 4.1.4. Software upgrades, etc Software upgrades will be made only by a service representative. The new version of the software will be noted in the maintenance log and all other appropriate locations. 4.1.5. Reference materials Covered above. 5. Analytical Procedures 5.1. This section will be used to address: 5.1.1. Procedure/analytical method 5.1.1.1 Blood. a. Remove one blood tube for analysis. b. Label two headspace vials with DR# (Departmental Record number)

and subject's name and vial number. c. Before preparing each case sample check that the DR# and name on

both vials correspond with DR# and name on blood tube. d. Dispense 2500 ?l n-propanol (0.015% w/v ISTD) and 250 ?l of sample

from blood tube into headspace vial. Repeat for second vial. Cap and crimp both vials. e. Place prepared vials in rack in front of blood tube. 5.1.1.2. Other liquids. a. Remove cap and determine if scent of liquid indicates alcohol. If not, the sample may be handled neat in the same way as a blood sample. If yes, then the sample should be diluted by a factor of 50 or 100 in purified water. b. Label two headspace vials with DR# and subject's name and vial number.

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SCOTTSDALE POLICE DEPARTMENT FORENSIC SERVICES DIVISION-

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Blood Alcohol Analysis Procedures Manual

Original Adoption Date: 2-22-10 Version: PM-TOX-001.1 Version Effective Date: 7-11-12 Issuing Authority: Kris Whitman, QAM

c. Before preparing each case sample check that the DR# and name on both vials correspond with DR# and name on sample tube.

d. Dispense 2500 ?l n-propanol (0.015% w/v ISTD) and 250 ?l of sample from container into headspace vial, cap and crimp. Repeat for second vial.

e. Place prepared vials in rack. 5.1.1.3. Calibration Standards.

a. Use the aqueous 0.02, 0.10, 0.20 and 0.40 % w/v ethanol standards made from a solution produced from 200 proof ethanol prepared as in 13.1.1 and verified against known traceable standards or equivalent commercial standards as verified in 4.1.3.1.1.

b. Label headspace vials with calibration standard level. c. Dispense 2500 ?l n-propanol (ISTD) and 250 ?l of calibration standard

into headspace vial, cap and crimp. 5.1.1.4. Control Standards Preparation: Blank, Control Reference Material

(CRM), Mixed and Check Standards preparation: a. Prepare single vials for the mixed standard, CRMs, Check Standards,

and blank in the same manner as samples and calibrators. 5.1.1.5. Place prepared vials into preassigned locations in the HS110

autosampler tray and continue analysis according to work instructions. 5.1.1.6. Capping. Capping of vials may be accomplished using a pneumatic

crimper or a hand crimper. Despite the best efforts of the analyst, a cap may appear to be crimped properly but not be making a gastight seal. A loose cap may be identified post analysis as a sample having an internal standard area count more than 25% lower than the average internal standard area count measured for the controls analyzed in that batch for that analytical day.

5.1.2. Sampling plan Not applicable. Sampling Procedure The sampling procedure for the analysis of whole blood or other liquid samples requires that the sample be homogeneous before removal of aliquots. This will be achieved by allowing all liquid samples to warm to room temperature, thoroughly mixing, and inspecting for clots. Clotted samples will be ground as necessary to homogenize. Sample Selection Any item for submitted for the same testing may be selected by the analyst either randomly or by evaluating volume or other physical properties which may make

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SCOTTSDALE POLICE DEPARTMENT FORENSIC SERVICES DIVISION-

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Blood Alcohol Analysis Procedures Manual

Original Adoption Date: 2-22-10 Version: PM-TOX-001.1 Version Effective Date: 7-11-12 Issuing Authority: Kris Whitman, QAM

one item preferable to another. The item selected will be documented in the notes. 5.1.3. Reagents Not applicable. 5.1.4. Quantification 5.1.4.1 Whole Blood: Prepare as described in the above and report as

"...analysis of the blood..." and the lower of the two replicate values. 5.1.4.2. Clotted Blood: Place contents of collection tube into a tissue grinder and

grind sample, then prepare as above. Report as "...analysis of the blood..." and the lower of the two replicate values. 5.1.4.3. Serum, Plasma: Separate supernatant from cellular material, if present. Prepare as above and report that sample is improper and no determination of blood alcohol concentration will be made. Report presence or absence of ethanol only. If a quantitative report must be issued, the analyst may either report the serum or plasma analyzed concentration and make it abundantly clear by the wording (e.g. use of all caps, and bold lettering) that the numerical value does not refer to whole blood. The analyst may also perform an appropriate conversion calculation and add it to the case notes. 5.1.4.4. Trace samples: Obtained values less than 0.020 BAC but greater than 0.010 BAC will be reported out as "Trace ethanol detected" or other similar phrasing. Samples where obtained value is less than 0.010 will be reported as "ethanol not detected". 5.1.4.5. Report the lower of the two case results truncated to three decimal places for the valid duplicate. 5.1.4.6. For liquid alcohol samples, the amount reported is the obtained value multiplied by the appropriate dilution factor. This number is reported as a % w/v as "...analysis of the liquid ..." 5.1.5. Controls and Standards (traceability) NIST traceable standards are used whenever they are commercially available. 5.1.6. Interpretation guidelines/uncertainty measurements Estimation of uncertainty for blood alcohol analysis was evaluated in accordance with ASCLD/LAB International requirements in conjunction with ARS statutes and was determined to be necessary. Full explanation and information may be found separately in the binder labeled as `SPD Blood Alcohol Uncertainty of Measurement". The general outline is as follows: 5.1.6.1 Previous estimation. A previous estimation of the SPD lab standard deviation of measurement for known alcohol standards had been determined to be 1 for years 2005-July 2009. This was determined

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SCOTTSDALE POLICE DEPARTMENT FORENSIC SERVICES DIVISION-

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Blood Alcohol Analysis Procedures Manual

Original Adoption Date: 2-22-10 Version: PM-TOX-001.1 Version Effective Date: 7-11-12 Issuing Authority: Kris Whitman, QAM

empirically from data gathered over the years and did not reflect a combined uncertainty as recommended by the ASCLD/LAB International guidelines. 5.1.6.2 Current Instrumentation Initial Estimation. 5.1.6.2.1. Purpose. In 2009, the HSGC instruments to be used in the SPD lab

were installed new in conjunction with the opening of the new facility. While the instruments were similar, they were not identical, as PerkinElmer had made upgrades in both software and firmware since the purchase of the original instrument. 5.1.6.2.2. Data. Only data collected on the new instruments was used to calculate the uncertainty of measurement which is now to be reflected. Quality control standards at all levels in whole blood matrix were used. Combined uncertainty was determined using the Root Sum Squares technique. The coverage factor was determined using the Student's t-table for n100 measurements to be 3.1 at a 99.7% confidence interval (CI). The combined uncertainty was determined to be 0.54. The expanded uncertainty (Ue)is the combined uncertainty (Uc) x k (k=3.2) and is equal to 1.7 for CI = 99.7. For a CI of 99.9999, k=5, and the Ue is 4.95. 5.1.6.5 Calculation. Using a standard normal distribution, a CI of 99.9999 can be obtained by multiplying Uc (0.54) by 5. As per the standard practice of the SPD lab, that would yield a scientific certainty that the true value for any sample within ?5% of the measured value. This is consistent with the practice of our laboratory and well within the AZDPS regulatory requirement that obtained values for known alcohol samples be within ? 10% for a permit to be issued. 5.1.6.6. Application. This uncertainty value may be reflected in a case file, in a report, or may be maintained in a file. 5.1.6.7. Reevaluation. Uncertainty will be observed on an ongoing basis but recalculated when a significant change is made to the procedure, instrumentation, or recommended method for calculation.

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